The South American perennial Stevia rebaudiana Bertoni is known for its natural sweeteners. The sweetness of the Stevia plant is attributed to the presence of high intensity sweet glycosides. The glycosides derived from Stevia (steviol glycosides) include stevioside (4-13% dry weight), Rebaudioside A (2-4%), Rebaudioside C (1-2%), dulcoside A (0.4-0.7%) and traces of steviolbioside, rubusoside and Rebaudiosides B, D, E, and F (Kinghorn (2002) Kinghorn (ed.) Stevia, the Genus Stevia. Medicinal and Aromatic Plants—Industrial Profiles, Vol. 19. Taylor and Francis, London and NY). Among these glycosides, only stevioside and Rebaudioside A are currently commercially available in large quantities. The raw materials for manufacturing stevioside and Rebaudioside A are the crushed leaves of the perennial shrub. Conventional extraction processes to obtain steviol glycoside-containing extracts that are described in the literature follow similar methodologies: stevia leaves are extracted with hot water or alcohols, the extract is clarified by precipitation with salt or alkaline solutions and concentrated, and the extract is re-dissolved in short alcohols (methanol or ethanol) for crystallization of the glycosides.
Methods for extracting Rebaudioside C have also been developed. WO 2011/037959 describes the purification of Rebaudioside C by refluxing a crude stevia extract solid or a crude Rebaudioside C solid in a mixed solvent (95% EtOH/MeOH/H2O=12.5/6/2), or (95% propanol/MeOH=12.5/7.5). The solution is subsequently cooled to 22° C. and seeded with 93-98% pure Rebaudioside C crystals. The resulting mixture is left at 22° C. for 16 hours and the white crystalline product is filtered, washed twice with an EtOH/MeOH (4/1) mixture and dried to yield purified Rebaudioside C product. The major drawback of this process is that high purity Rebaudioside C is needed to induce purification. In addition, the yield of this process and the purity of the obtained material are not disclosed.
Chinese Patent No. 102127130 describes a method of purifying Rebaudioside C by passing a crude solution containing about 17% of Rebaudioside C through an ultra-filtration membrane to generate a fraction having a 26% Rebaudioside C content. The fraction is concentrated at 55° C. until the solid content reaches 40%. Subsequently, the liquid and solid are separated such that the solid contains about 33% Rebaudioside C. This crude Rebaudioside C is further purified by recrystallization from a mixed solvent (87% EtOH/68% MeOH/87% acetone=3/2/1) and the resulting purity of Rebaudioside C is 85.4%.
Conventional methods to produce and purify Rebaudioside C require numerous reaction steps or iterative purification steps. There is a need for a simple, efficient, and economical method for production of high purity Rebaudioside C, which can be used in food, beverage, pharmaceutical, cosmetic and other industries.